About ten million Americans, predominantly women, suffer from dry eye disease, arising from a variety of causes including aging, refractive surgery, menopause, autoimmune disease, and trauma. Although the etiology of each of these conditions differs, a common feature of all is a decrease in lacrimal gland secretion and damage to the ocular surface resulting in inflammation, irritation, and pain. Current treatments are only palliative as they target the ocular surface, but not the cause of the disease, a decrease in lacrimal gland secretion. Most individuals with dry eye disease could benefit from improved protein, electrolyte, and water secretion from the lacrimal gland. In dry eye lacrimal gland cells are diminished by apoptosis or necrosis, thus restoration of diseased lacrimal gland using exogenous progenitor cells or stimulating endogenous regeneration would improve the health of the ocular surface by replacing lost cells and could cure dry eye. Our laboratory is the first to develop a reproducible protocol to isolate from adult, rat exorbital lacrimal gland progenitor cells that could be used to repair the diseased lacrimal gland. This finding suggests that stem cells or cells capable of regeneration are present in the lacrimal gland and can be activated to repair damaged tissue. The regeneration of the three differentiated cells types of the lacrimal gland, acinar, duct, and myoepithelial cells, must occur via one of three mechanisms. The newly formed differentiated cells are derived from proliferation of cells of the same cell type, derived from proliferation of cells of a different cell type or derived from immature, undifferentiated, progenitor cells. The overall goal of the project is to repair the lacrimal gland, restore its secretory function, and develop a new strategy to cure dry eye by using adult lacrimal gland progenitor cells. In preliminary data we found that a small number of lacrimal gland cells were positive for stem cell markers and could be reproducibly isolated. We hypothesize that the adult lacrimal gland contains a small population of progenitor cells and that these cells can be induced either in vitro or in vivo to differentiate into functioning lacrimal gland nerve, acinar, duct, and myoepithelial cells that can repair the gland. To achieve these goals, we will use a panel of stem cell markers for immunohistochemistry, western blotting, and flow cytometry to identify the progenitor cells in culture and compare these markers with their localization in the in vivo lacrimal gland. The progenitor cell niche will be determined by double labeling with antibodies selective for the major cell types of the lacrimal gland to determine which cell type is needed for progenitor cell formation. We will then label proliferating cells from injured mouse lacrimal glands with bromodeoxyuridine to follow cell lineages during regeneration. We will also use lineage tracing using GFAP and Ascl3 transgenic mice. Using a protocol similar to one that forms neurospheres, we will determine if lacrimal gland progenitor cells in culture form floating spheres that can be used for repair. PUBLIC HEALTH RELEVANCE: Millions of Americans suffer from dry eye disease. The causes are varied and include aging, refractive surgery, menopause, autoimmune disease, and trauma causing damage to the ocular surface resulting in pain, inflammation, and irritation. There is no cure for dry eye and as the lacrimal gland is the primary contributor to the aqueous layer of the tear film, replacement of a diseased lacrimal gland using stem cells would improve the health of the ocular surface.